1,8-Naphthyridines VIII. Novel 5-aminoimidazo[1,2-a] [1,8]naphthyridine-6-carboxamide and 5-amino[1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamide derivatives showing potent analgesic or anti-inflammatory activity, respectively, and completely devoid of acute gastrolesivity

Article abstract of DOI:10.1016/j.ejmech.2009.10.020

On the basis of the very interesting pharmacological properties shown by the 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 1, previously described by us, we have now prepared the 5-aminoimidazo[1,2-a][1,8]naphthyridine-6-carboxamide derivatives 2a-o (a new structural class) whose tricyclic system is isosteric to that of compounds 1. Both compounds 2 and some new properly substituted compounds 1 (1f-k) now synthesized were tested in vivo for their analgesic and anti-inflammatory activities: on the whole, compounds 2 showed notable analgesic properties, whereas many compounds 1 exhibited a very potent anti-inflammatory activity, coupled to scarce analgesic activity. All the effective compounds proved to be completely devoid of acute gastrolesivity (gastric damage) in rats (at the 200 mg kg^-1 oral dose).

Full text of DOI:10.1016/j.ejmech.2009.10.020

European Journal of Medicinal Chemistry 45 (2010) 352–366
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,8-Naphthyridines VIII. Novel 5-aminoimidazo[1,2-a]
[1,8]naphthyridine-6-carboxamide and 5-amino[1,2,4]triazolo[4,3-a]
[1,8]naphthyridine-6-carboxamide derivatives showing potent analgesic
or anti-inflammatory activity, respectively, and completely devoid of
acute gastrolesivity
,
a
a
a
b
Giorgio Roma a , Mario Di Braccio , Giancarlo Grossi , Daniela Piras , Vigilio Ballabeni ,
*
Massimiliano Tognolini b, Simona Bertoni b, Elisabetta Barocelli b
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Genova, Viale Benedetto XV, 3, I-16132 Genova, Italy
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita di Parma, Viale G.P. Usberti 27/A, I-43100 Parma, Italy
b
`
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of the very interesting pharmacological properties shown by the 5-amino[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide derivatives 1, previously described by us, we have now prepared
the 5-aminoimidazo[1,2-a][1,8]naphthyridine-6-carboxamide derivatives 2a–o (a new structural class)
whose tricyclic system is isosteric to that of compounds 1. Both compounds 2 and some new properly
substituted compounds 1 (1f–k) now synthesized were tested in vivo for their analgesic and anti-
inflammatory activities: on the whole, compounds 2 showed notable analgesic properties, whereas many
compounds 1 exhibited a very potent anti-inflammatory activity, coupled to scarce analgesic activity. All
the effective compounds proved to be completely devoid of acute gastrolesivity (gastric damage) in rats
(at the 200 mg kgꢀ1 oral dose).
Received 10 July 2009
Received in revised form
22 September 2009
Accepted 9 October 2009
Available online 13 November 2009
Keywords:
Imidazo[1,2-a][1,8]naphthyridines
[1,2,4]Triazolo[4,3-a][1,8]naphthyridines
Anti-inflammatory
Ó 2009 Elsevier Masson SAS. All rights reserved.
Analgesic
Gastric damage
Structure–activity relationships
1. Introduction
action of this activity [4], whereas an in vitro study proved that the
anti-inflammatory activity of the most potent compound 1b is
In previous papers [1–4] we described the synthesis and the
often remarkable anti-inflammatory and/or analgesic properties of
new tricyclic compounds which proved to be devoid of acute gas-
trolesivity and toxicity. In particular, a number of 5-amino[1,2,4]-
triazolo[4,3-a][1,8]naphthyridine-6-carboxamides 1 proved to be
potent analgesic and/or anti-inflammatory agents, depending on
the substituents (Table 1) [1,3,4]. It is interesting to point out that
the notable potency difference between the analgesic and the anti-
inflammatory activities of nearly each effective compound 1 (see
Table 1) suggested that the mechanisms of action supporting the
two activities are different [4].
based on its ability to counteract polymorphonuclear cells adhesion
to endothelial cells and its inhibition of both superoxide anion
production and myeloperoxidase release, without involving the
cyclooxygenase (COX) inhibition [5].
On the basis of the pharmacological results previously obtained
by us (see the most significant ones in Table 1), we have now
pursued our studies aimed at synthesizing further properly
substituted compounds 1 and their isosteric analogues 5-amino-
imidazo[1,2-a][1,8]naphthyridine-6-carboxamides 2 (Fig. 1), and at
evaluating their pharmacological properties. The now synthesized
compounds 1f–k and 2a–o have been therefore tested for their
analgesic (in mice writhing test) and anti-inflammatory (in rat paw
edema test) activities, and for their acute gastrolesivity in rats. The
compounds 1 and 2 endowed with statistically significant anti-
nociceptive activity have been also tested for their inhibition of
spontaneous locomotor activity in mice.
Actually, the very potent analgesic compound 1e was studied
through a number of tests without discovering the mechanism of
* Corresponding author. Tel.: þ39 10 353 8374; fax: þ39 10 353 8358.
E-mail address: roma@unige.it (G. Roma).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.020

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
353
Table 1
Structures and pharmacological data of the most potent anti-inflammatory (1a–c) and analgesic (1d, e) agents previously described by us
(CH3)2CH
N
N
N
N
CON(C2H5)2
III
IV
R
R
N
1a-e
Compound
Dose
Analgesic
Anti-inflammatory
activityb, f
(% inhibition)
Spontaneous mice locomotor
activityc, e
(% inhibition)
Acute gastrolesivity
in ratsd, e
RIII
RIV
(mg kgꢀ1 oral)
activitya, e
(% inhibition)
N
1a
1b
NH–CH(CH3)2
100
50
25
83** g
54**
33*
0
81**
75**
75**
68**
98**
64*
26
0/8
12.5
0
NH–i–C4H9
100
50
25
12.5
6.25
100**
33
–
–
–
70**
66**
61**
50**
27*
18
–
–
–
–
0/8
100
50
25
12.5
6.25
81**
59**
17
–
71**
67**
65**
51*
53*
22
–
–
–
CH3
N
1c
0/8
0/8
C2H5
–
42*
1d
NH–CH(C2H5)2
100
50
25
12.5
6.25
91**
75**
58**
49*
50**
47**
47**
45*
97**
84**
70**
92**
81**
36*
11
100
50
25
12.5
6.25
3.12
1.6
88**
85**
90**
92**
83**
85**
39*
23
–
–
–
–
94**
100**
99**
74**
19
N
NCH3
1e
0/8
–
–
3
–
a
Acetic acid induced writhing in mice.
Carrageenan induced rat paw edema.
Activity counted in 30 min in mice.
Number of rats showing gastric lesions.
b
c
d
e
f
Data from Ref. [3] and Ref. [4] for compounds 1b, e (oral dose 100 mg kgꢀ1) and 1a, c, d (oral dose 200 mg kgꢀ1), respectively.
Data from Ref. [1] for compound 1b, from Ref. [4] for 1a, c, d.
g
*P < 0.05, **P < 0.01 significance as compared to controls (Student’s t-test).
2. Chemistry
afterremovalofethanolandexcess1-methylpiperazineinvacuo, was
heated as a crude compound in phosphorus oxychloride at reflux for
The new 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-car-
boxamide derivatives 1f–k were prepared through the synthetic
routes reported in Scheme 1. Thus, the reaction of 2-aminonicotinic
acid with ethyl N-ethyl-N-methylmalonamate [6] in the presence of
phosphorus oxychloride (110 ꢁC, 3 h)affordeda moderateyieldof the
novel 4-chloro-N-ethyl-N-methyl-1,2-dihydro-2-oxo-1,8-naphthyr-
idine-3-carboxamide 3 which was then heated in refluxing phos-
phorus oxychloride for 45 min, to give a high yield (88%) of the
corresponding 2,4-dichloroderivative 4a. Besides, the methyl 1,2-
R
R
9
N8
N7
8
9
1
N
1
N
7
N
N
N
2
3
2
3
6
6
RI
RI
CON
CON
5
4
5
4
RII
RII
III
III
R
R
N
N
IV
IV
R
R
2
dihydro-4-hydroxy-2-oxo-1,8-naphthyridine-3-carboxylate
6 [7]
1
washeatedinaclosedvessel(160 ꢁC,16 h)withanethanolsolutionof
Fig. 1. Structures of the substituted 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamides 1 and 5-aminoimidazo[1,2-a][1,8]naphthyridine-6-carboxamides 2.
excess 1-methylpiperazine to give the corresponding amide 7 which,

354
G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
Scheme 1. Synthetic routes to the 9-alkyl-5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 1f–k.
2 h to give a 41% overall yield of the 2,4-dichloroderivative 4b. From
the subsequent cyclocondensation of the dichloroderivatives 4a and
4b with isobutyrohydrazide (Dowtherm A,160 ꢁC, 30 min), the novel
5-chloro[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide
derivatives5aand5b, respectively, were obtained inmoderateyields.
Finally, the treatment of the proper 5-chloroderivatives 5a,b, 5c [4] or
5d [3] with an excess of suitable amines (closed vessel, 140 ꢁC, 16 h)
afforded high yields of the desired compounds 1h, 1k, 1g, 1i,
respectively, or of the intermediate 1l (from 5d). The 5-(dia-
lkylamino)derivatives 1j and 1f were then obtained (in good yields)
by methylation of the novel compound 1l or of the previously
described 1m [1], respectively, using iodomethane in 2-butanone at
reflux (1.5 h) in the presence of anhydrous K2CO3/KOH.
The novel 5-aminoimidazo[1,2-a][1,8]naphthyridine-6-carbox-
amide derivatives 2a–o were obtained through the synthetic routes
described in Schemes 2 and 3. First, the 2,4-dichloroderivative 4c
[8] was treated in anhydrous ethanol at room temperature with an
excess of (2,2-dimethoxyethyl)amine (2 h) or propargylamine
(24 h) to give good yields of a nearly equimolar mixture of the
regioisomers 8a and 9a or 8b and 9b, respectively. Compounds 8a
and 8b were easily separated from the corresponding isomers 9a
and 9b by column chromatography. The cyclocondensation or the
cycloaddition, respectively, of the substituted 2-aminoderivatives
8a and 8b, heated at 180 ꢁC in Dowtherm A for 20 min, in the
presence of monohydrate p-toluenesulphonic acid, afforded then
high yields of the substituted 5-chloroimidazo[1,2-a][1,8]naph-
thyridine-6-carboxamides 10a or 10b, respectively. The treatment
of 5-chloroderivatives 10a or 10b with proper amines in excess,
under severe conditions (heating at 160 ꢁC in closed vessel, 16 h),
gave good yields (60–82%) of the desired 5-aminoderivatives 2a,
c–h, j–l. On the other hand, the 5-(isobutylamino)derivatives 2b
and 2i were prepared starting from the previously described
2-chloro-N,N-diethyl-4-(isobutylamino)-1,8-naphthyridine-3-car-
boxamide 9c [9]. Thus, the reaction of 9c with propargylamine (in
Dowtherm A at 180 ꢁC for 20 min, in the presence of monohydrate
p-toluenesulphonic acid) afforded directly the 5-amino-9-methyl-
imidazo[1,2-a][1,8]naphthyridine-6-carboxamide derivative 2i in
good yield. The reaction of 9c with (2,2-dimethoxyethyl)amine
(ethylene glycol, 140 ꢁC, 2 h) afforded a good yield of the 2,4-dia-
minoderivative 11a, whose cyclocondensation (90% yield) to the
desired imidazo[1,2-a][1,8]naphthyridine derivative 2b was
obtained by heating compound 11a in Dowtherm A at 180 ꢁC for

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
355
Scheme 2. Synthetic routes to the 5-amino-N,N-diethylimidazo[1,2-a][1,8]naphthyridine-6-carboxamide derivatives 2a–l.
20 min, in the presence of monohydrate p-toluenesulphonic acid
(Scheme 2).
At last, the N,N-diethyl-5-(4-methyl-1-piperazinyl)imidazo[1,2-
a][1,8]naphthyridine-6-carboxamide derivatives 2m–o were
obtained starting from the glycine ethyl ester derivative 8c, previ-
ously described by us [9]. As first step, the reaction of 8c with excess
1-methylpiperazine in dimethyl sulphoxide (130 ꢁC, 3 h) afforded
a 78% yield of the substituted 2,4-diamino-1,8-naphthyridine-3-

356
G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
Scheme 3. Synthetic routes to the N,N-diethyl-5-(4-methyl-1-piperazinyl)imidazo[1,2-a][1,8]naphthyridine-6-carboxamide derivatives 2m–o.
carboxamide 11b, which was the common intermediate for the
synthesis of the three compounds 2m–o. Thus, the cyclo-
condensation of 11b in a mixture of polyphosphoric acid-phos-
phorus oxychloride (120 ꢁC, 3.5 h) afforded a 55% yield of the
tricyclic 9-ethoxyderivative 2m, while the reaction of 11b with
excess N,N-dimethylformamide diethyl acetal at reflux (120 ꢁC,
24 h) afforded the tricyclic 8-ethoxycarbonyl derivative 2o. Finally,
compound 11b was treated with an excess of ethanolic solution of
dimethylamine (closed vessel, 150 ꢁC, 16 h) to give (84%) the N,N-
dimethylglycinamide derivative 11c which, in turn, cyclocondensed
in a mixture of polyphosphoric acid-phosphorus oxychloride
(110 ꢁC, 30 min) to afford (68%) the tricyclic 9-(dimethylamino)-
derivative 2n (Scheme 3).
dose [3] (see Section 5.2.). The above pharmacological results are
listed in Table 2.
The new substituted 5-amino[1,2,4]triazolo[4,3-a][1,8]naph-
thyridine-6-carboxamides 1f–k herein described have been
designed to obtain further interesting anti-inflammatory agents,
taking account of our previous results in this structural and phar-
macological field. In actual fact, except for 1k and partially for 1h,
all the compounds 1 now synthesized showed an effective anti-
inflammatory activity (carrageenan induced rat paw edema assay).
The compounds 1g, i, j exhibited significant anti-inflammatory
activity down to the 3.12, 12.5, and 12.5 mg kgꢀ1 oral doses (edema
inhibition: 35% P < 0.05, 32% P < 0.01, and 44% P < 0.01, respec-
tively). It is interesting to point out that, within this series, 1g has
proved to be the most potent anti-inflammatory agent until now
synthesized by us (40% edema inhibition, P < 0.01 and 35% edema
inhibition, P < 0.05, at the 6.25 and 3.12 mg kgꢀ1 oral doses,
respectively), whereas compound 1j was the most effective one,
producing maximal edema inhibition (71% P < 0.01, at the
100 mg kgꢀ1 dose).
The structures attributed to the new compounds 1 and 2
herein described are in accordance with the results of elemental
analyses, and their IR and 1H NMR spectral data (see Table 3 and
Section 5.1).
3. Results and discussion
As regards the antinociceptive activity shown by compounds
1f–k (acetic acid induced writhing test in mice), they all
exhibited an evident significant activity only at the 100 mg kgꢀ1
dose (compounds 1f–j, inhibition range 71–92% P < 0.01;
compound 1k, inhibition 48% P < 0.05). Actually, five compounds
(1f–j) out of six significantly inhibited at the 100 mg kgꢀ1 dose
(inhibition range 48–96%) the spontaneous locomotor activity of
the treated mice.
As concerns the 5-aminoimidazo[1,2-a][1,8]naphthyridine-6-
carboxamide derivatives 2a–o, compounds 2b, c, j, k showed
statistically significant analgesic activity down to the 25 mg kgꢀ1
dose and 2m down to the 12.5 mg kgꢀ1 dose (inhibition range:
71–97% at the 100 mg kgꢀ1 dose, 48–83% at the 25 mg kgꢀ1
dose). Compounds 2a, d–h, n, o exhibited a significant analgesic
activity only at the 100 mg kgꢀ1 dose (inhibition range 41–83%).
Actually, compounds 2b, c, j, k, m appear to be very effective
antinociceptive agents: the most potent (48% inhibition at
the 12.5 mg kgꢀ1 dose, P < 0.05) and most effective of them
The analgesic and anti-inflammatory activities of compounds
1f–k and 2a–o (Table 2) have been evaluated in vivo at the initial
dose of 100 mg kgꢀ1 oral. The compounds exhibiting a statistically
significant activity at this dose were further tested at doses
decreasing by a factor of two, until statistically significant activity
was no longer observed. The compounds showing significant
analgesic activity in writhing test were evaluated (at properly
chosen doses) for their effect on spontaneous locomotor activity in
mice, after oral administration, in order to verify the occurrence of
confounding sedative effect. The compounds endowed with
significant analgesic and/or anti-inflammatory activities were also
tested for their acute gastrolesivity in rats (at the 200 mg kgꢀ1 oral
dose): for all the compounds tested no acute gastrolesive effect was
detected in each of the eight treated rats (0/8), whereas indo-
methacin, used as reference anti-inflammatory drug, caused gastric
ulcers in seven of the eight treated rats (7/8) at the 10 mg kgꢀ1 oral

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
357
Table 2
Structures and pharmacological data of compounds 1f–k and 2a–o
R
R
N
N
N
N
N
N
N
N
N
RI
RI
CON
CON
RII
RII
III
IV
III
R
R
R
IV
1f-k
R
2a-o
Dose
Analgesic activitya Anti-inflammatory
Spontaneous mice
Acute gastrolesivity
RI
R
III
Compd.
R
(mg kgꢀ1 (% inhibition)
oral)
activityb (% inhibition) locomotor activityc in ratsd
(% inhibition)
N
N
RIV
RII
92**e
26
–
39**
42**
28*
8
60**
0/8f
100
50
25
CH3
–
–
–
N
N
1f
C2H5
N(C2H5)2
CH2CH(CH3)2
12.5
–
0/8
100
50
25
12.5
6.25
3.12
1.6
81**
21
–
–
–
60**
50**
47**
49**
40**
35*
48*
–
–
–
–
H
N
C2H5
H
1g
CH(CH3)2
CH2CH(CH3)2
–
–
–
–
18
100
50
82**
0
46*
0
96**
–
0/8
0/8
CH3
H
N
N
1h
1i
CH(CH3)2
CH(CH3)2
C2H5
CH(CH3)2
100
50
25
12.5
6.25
79**
28
–
–
–
60**
49**
52**
32**
25
64**
H
–
–
–
–
N
N(C2H5)2
CH CH CH
2
2
100
50
25
12.5
6.25
71**
37
–
–
–
71**
55**
42**
44**
23
62**
0/8
0/8
CH3
–
–
–
–
N
1j
CH(CH3)2
N(C2H5)2
CH2CH2CH3
100
50
48*
29
0
–
0
–
H
N
N
NCH3
1k
2a
2b
CH(CH3)2
CH2CH(CH3)2
100
50
25
60*
2
–
29*
26*
25*
0
89**
0/8
0/8
H
N
C2H5
–
–
–
H
H
N(C2H5)2
N(C2H5)2
12.5
–
100
50
25
71**
48**
48*
0
0
–
–
–
68**
28
–
H
N
CH2CH(CH3)2
12.5
–
100
50
25
80**
74**
56*
32
0
–
–
–
77**
41*
33
0/8
0/8
0/8
N
NCH3
2c
2d
2e
H
H
H
N(C2H5)2
N(C2H5)2
N(C2H5)2
12.5
–
100
50
80**
30
5
–
67**
–
N
NC2H5
100
41*
12
0
–
39*
–
N
NCH2CH2OH 50
(continued on next page)

358
G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
Table 2 (continued)
Dose
Analgesic activitya Anti-inflammatory
Spontaneous mice
Acute gastrolesivity
RI
R
III
Compd.
R
(mg kgꢀ1 (% inhibition)
oral)
activityb (% inhibition) locomotor activityc in ratsd
(% inhibition)
N
N
RIV
RII
0/8f
100
50
46*,e
0
26*
10
42
–
N
NCH2
2f
H
N(C2H5)2
N(C2H5)2
100
50
63**
0
16
–
86**
–
0/8
N
N
2g
H
CH3O
100
50
73**
0
38**
20
55**
–
0/8
H
N
CH3
2h
2i
9-CH3
9-CH3
N(C2H5)2
H
N
N(C2H5)2
100
0
0
–
–
CH2CH(CH3)2
100
50
25
85**
86**
83**
21
0
–
–
–
80**
20
–
0/8
N
NCH3
2j
9-CH3
9-CH3
9-CH3
N(C2H5)2
N(C2H5)2
N(C2H5)2
12.5
–
100
50
25
83**
48**
54*
0
10
–
–
63**
46*
58**
–
0/8
N
NC2H5
2k
2l
12.5
–
N
NCH2
100
17
0
–
–
100
50
25
12.5
6.25
97**
62**
54**
48*
17
–
–
–
–
60**
46**
53**
15
0/8
N
NCH3
2m
9-OC2H5
N(C2H5)2
26
–
100
50
83**
16
0
–
44**
30
0/8
0/8
N
NCH3
2n
2o
9-N(CH3)2
N(C2H5)2
100
50
75**
2
0
–
0
–
N
NCH3
8-COOC2H5 N(C2H5)2
Indomethacin
Diazepam
10
10
84**g
–
51**g
–
–
7/8h
–
98**h
a
Acetic acid induced writhing in mice.
Carrageenan induced rat paw edema.
Activity counted in 30 min in mice.
Number of rats showing gastric lesions.
*P < 0.05, **P < 0.01 significance as compared to controls (Student’s t-test).
The compounds 1f–k, 2a–o were tested at the 200 mg kgꢀ1 oral dose.
Data from Ref. [2].
b
c
d
e
f
g
h
Data from Ref. [3].
(97% inhibition at the 100 mg kgꢀ1 dose, P < 0.01) was 2m,
whose 5-(4-methyl-1-piperazinyl) proved to be the most effec-
tive 5-amino substituent for their analgesic activity, as we
previously remarked [3] in the case of compounds 1 (Table 1,
compound 1e).
It must be pointed out that the lower analgesic activity shown
by compound 2c, with respect to compounds 2j, m, seems clearly to
indicate that the presence of proper 9-substituents has been useful
for such their activity. In this connection, we attempted to obtain
the 9-CH(CH3)2 substituted derivative of 2c (see the structure of the
potent analgesic agent 1e), but until now it was not obtainable. On
the other hand, the introduction of the isosteric 9-N(CH3)2
substituent was ineffective (see compound 2n, Table 2). Concerning
then the possible connection between analgesia and sedation,
compounds 2a–h, j, k, m, n, at the 100 mg kgꢀ1 oral dose, showed
significant inhibition for both the analgesic and spontaneous mice
locomotor activities (inhibition ranges 41–97% and 39–89%,
respectively). Compounds 2c, k, m showed also, down to lower
doses, statistically significant inhibitions for both these activities.
As regards the anti-inflammatory activity, it resulted to be
completely or almost absent in twelve of the fifteen compounds
2a–o (Table 2).

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
359
Table 3
Physical and chemical data of compounds 1f–k and 2a–o
R
R
N
N
N
N
N
N
N
N
RI
RI
CON
CON
RII
RII
III
IV
III
IV
R
R
R
N
1f-k
R
2a-o
Compd.a
Yield (%)
75
M.p. (ꢁC), solventb
Molecular
Formulac
IRd (cmꢀ1
)
1H NMRe
(d, ppm)
1f
142–144 (A)
C21H30N6O
1640 s (CO), 1601,
0.94 and 0.95 [2d, 6H, NCH2CH(CH3)2], 1.26 and 1.32 [2 t, 3H þ 3H,
N(CH2CH3)2], 1.49 (t, 3H, 9-CH2CH3), 2.04 [m, 1H, NCH2CH(CH3)2],
2.68 [m, 1H, 1H of NCH2CH(CH3)2], 2.88 (s, 3H, NCH3), 3.11 [m, 1H,
1H of NCH2CH(CH3)2], 3.25–3.72 [m, 5H, 3H of N(CH2CH3)2 þ 9-
CH2CH3], 3.90 [m, 1H, 1H of N(CH2CH3)2], 7.50 (dd, 1H, H-3), 8.49
(dd, 1H, H-4), 8.68 (dd, 1H, H-2).
1586, 1554, 1522 w.
1g
1h
1i
78
96
95
81
91
129–131 (A)
176–177 (B)
174,5–176 (B)
169–170 (C)
227–228 (B)
C19H26N6O
3220 br (NH), 1615 s
(CO), 1575, 1557,
1521 w.
1.02 [d, 6H, NCH2CH(CH3)2], 1.29 (t, 3H, NCH2CH3), 1.53 [d, 6H,
9-CH(CH3)2], 2.00 [m, 1H, NCH2CH(CH3)2], 3.32–3.59 [m, 4H,
NCH2CH(CH3)2 þ NCH2CH3], 4.41 [m, 1H, 9-CH(CH3)2], 7.46 (dd, 1H,
H-3), 8.52 (dd, 1H, H-4), 8.70 (dd, 1H, H-2), 10.53f and 10.71f (2 br
s, 1H þ 1H, NH þ NHCO).
C19H26N6O
3278 (NH), 1611 s,
br (CO), 1537.
1.19 and 1.27 (2 t, 3H, NCH2CH3), 1.30 [2d overlapped, 6H,
NCH(CH3)2], 1.50 and 1.53 [2d, 6H, 9-CH(CH3)2], 3.00 and 3.16 (2s,
3H, NCH3), 3.23–3.95 [m, 3H, NCH2CH3 þ NCH(CH3)2], 4.37 [m, 1H,
9-CH(CH3)2], 4.80f (br s, 1H, NH), 7.42 (dd, 1H, H-3), 8.36 (dd, 1H,
H-4), 8.63 (dd, 1H, H-2).
C20H26N6O
3267 (NH), 1615 s,
br (CO), 1549, 1518 w.
1.16 and 1.32 [2 t, 3H þ 3H, N(CH2CH3)2], 1.38 and 1.59 [2d,
3H þ 3H, 9-CH(CH3)2], 3.24–3.84 [m, 4H, N(CH2CH3)2], 3.95–4.12
(m, 2H, NCH2CH ¼ CH2), 4.17 [m, 1H, 9-CH(CH3)2], 5.18–5.40 (m,
2H, NCH2CH ¼ CH2), 5.95–6.17 (m, 1H, NCH2CH ¼ CH2), 6.20f (br s,
1H, NH), 7.03 (dd, 1H, H-3), 8.25 (dd, 1H, H-4), 8.40 (dd, 1H, H-2).
0.91 (t, 3H, NCH2CH2CH3), 1.25 and 1.32 [2 t, 3H þ 3H, N(CH2CH3)2],
1.48 and 1.56 [2d, 3H þ 3H, 9-CH(CH3)2], 1.70 (m, 2H, NCH2CH2CH3),
2.89 (s, 3H, NCH3), 2.92–3.85 [m, 6H, N(CH2CH3)2 þ, NCH2CH2CH3],
4.42 [m, 1H, 9-CH(CH3)2], 7.48 (dd, 1H, H-3), 8.43 (dd, 1H, H-4),
8.65 (dd, 1H, H-2).
0.99 and 1.01 [2d, 6H, NCH2CH(CH3)2], 1.34 and 1.62 [2d, 3H þ 3H,
9-CH(CH3)2], 2.01 [m, 1H, NCH2CH(CH3)2], 2.45 (s, 3H, NCH3),
2.50–3.90 [m, 10H, piperazine CH2’s þ NCH2CH(CH3)2], 4.14 [m, 1H,
9-CH(CH3)2], 6.27f (near d, 1H, NH), 6.91 (dd, 1H, H-3), 8.11 (dd, 1H,
H-4), 8.30 (dd, 1H, H-2).
1j
C21H30N6O
1629 s (CO), 1598,
1582, 1547, 1516 w.
1k
C22H31N7O
3267 (NH), 1606 s,
br (CO), 1549, 1522 w.
2a
2b
78
90
157–158 (C)
188–189 (D)
C17H21N5O
3263 (NH), 1606 s,
br (CO), 1540, 1517 w.
1.13, 1.30 and 1.34 [3 t, 3H þ 3H þ 3H, NCH2CH3 þ N(CH2CH3)2],
2.99–3.60 [m, 5H, NCH2CH3 þ 3H of N(CH2CH3)2], 4.08 [m, 1H, 1H
of N(CH2CH3)2], 5.30f (br s, 1H, NH), 6.94 (dd, 1H, H-3), 7.33 (d, 1H,
H-8), 7.96 (d, 1H, H-9), 8.14 (dd, 1H, H-4), 8.44 (dd, 1H, H-2).
1.04 [d, 6H, NCH2CH(CH3)2], 1.16 and 1.32 [2 t, 3H þ 3H, N(CH2CH3)2],
2.05 [m, 1H, NCH2CH(CH3)2], 2.84 [m, 1H, 1H of NCH2CH(CH3)2],
3.21–3.50 [m, 4H, 1H of NCH2CH(CH3)2 þ 3H of N(CH2CH3)2], 4.14 [m,
1H, 1H of N(CH2CH3)2], 5.35f (br s, 1H, NH), 7.01 (dd, 1H, H-3), 7.36 (d,
1H, H-8), 8.00 (d, 1H, H-9), 8.19 (dd, 1H, H-4), 8.48 (dd, 1H, H-2).
1.17 and 1.37 [2 t, 3H þ 3H, N(CH2CH3)2], 2.22–3.56 [m, 11H,
piperazine CH2’s þ 3H of N(CH2CH3)2], 2.40 (s, 3H, NCH3), 4.05 [m, 1H,
1H of N(CH2CH3)2], 7.46 (dd, 1H, H-3), 7.57 (d, 1H, H-8), 8.33 (d, 1H, H-
9), 8.48 (dd, 1H, H-4), 8.68 (dd, 1H, H-2).
1.11 (t, 3H, NCH2CH3), 1.14 and 1.34 [2 t, 3H þ 3H, N(CH2CH3)2], 2.23–
3.60 [m, 11H, piperazine CH2’s þ 3H of N(CH2CH3)2], 2.49 (q, 2H,
NCH2CH3), 4.01[m, 1H, 1H of N(CH2CH3)2], 7.42 (dd, 1H, H-3), 7.54 (d,
1H, H-8), 8.30 (d, 1H, H-9), 8.46 (dd, 1H, H-4), 8.65 (dd, 1H, H-2).
1.17 and 1.34 [2 t, 3H þ 3H, N(CH2CH3)2], 2.40–3.74 [m, 16H, piperazine
CH2’s þ 3H of N(CH2CH3)2 þ NCH2CH2OH; 15H after treatment with
D2O], 4.02 [m, 1H, 1H of N(CH2CH3)2], 7.44 (dd, 1H, H-3), 7.55 (d, 1H,
H-8), 8.32 (d,1H, H-9), 8.47 (dd, 1H, H-4), 8.66 (dd, 1H, H-2).
1.15 and 1.34 [2 t, 3H þ 3H, N(CH2CH3)2], 2.27–3.80 [m, 11H, piperazine
CH2’s þ 3H of N(CH2CH3)2], 3.60 (s, 2H, NCH2C6H5), 4.05 [m, 1H, 1H of
N(CH2CH3)2], 7.18–7.58 (m, 6H, H-3 þ C6H5), 7.55 (d, 1H, H-8), 8.31
(d,1H, H-9), 8.48 (dd, 1H, H-4), 8.65 (dd, 1H, H-2).
C19H25N5O
3264 (NH), 1600 s (CO),
1543, 1518 w.
2c
2d
2e
2f
74
78
77
77
82
183.5–184 (B)
147–148 (C)
187–188 (B)
175–176 (B)
217–218 (B)
C20H26N6O
C21H28N6O
C21H28N6O2
C26H30N6O
C26H30N6O2
1642 s (CO), 1598,
1582, 1541, 1514 w.
1629 s (CO), 1600,
1584, 1547, 1516 w.
3269 (OH), 1638 s (CO),
1601, 1585, 1544,
1518 w.
1633 s (CO), 1599,
1582, 1545, 1514 w.
2g
1635 s (CO), 1602,
1585, 1548, 1499.
1.17 and 1.37 [2 t, 3H þ 3H, N(CH2CH3)2], 2.78–3.65 [m, 11H, piperazine
CH2’s þ 3H of N(CH2CH3)2], 3.85 (s, 3H, OCH3), 4.09 [m, 1H, 1H of
N(CH2CH3)2], 6.77–7.08 (m, 4H, NC6H4OCH3), 7.45 (dd, 1H, H-3), 7.56
(d, 1H, H-8), 8.33 (d,1H, H-9), 8.54 (dd, 1H, H-4), 8.68 (dd, 1H, H-2).
(continued on next page)

360
G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
Table 3 (continued)
Compd.a
Yield (%)
M.p. (ꢁC), solventb
193–195 (D)
Molecular
Formulac
IRd (cmꢀ1
)
1H NMRe
(d, ppm)
2h
60
74
C17H21N5O
3293 (NH), 1611 s,
br (CO), 1539, 1512 w.
1.12 and 1.29 [2 t, 3H þ 3H, N(CH2CH3)2], 2.76 (near s, 3H, 9-CH3), 3.00
(s, 3H, NCH3), 3.18–3.50 [m, 3H, 3H of N(CH2CH3)2], 4.11 [m, 1H, 1H of
N(CH2CH3)2], 6.92f (br s, 1H, NH), 6.75 (dd, 1H, H-3), 6.95 (near s, 1H, H-
8), 8.01 (dd, 1H, H-4), 8.26 (dd, 1H, H-2).
2i
236–238 (E)
C20H27N5O
3266 (NH), 1598 s,
br (CO), 1545, 1510 w.
1.03 [d, 6H, NCH2CH(CH3)2], 1.13 and 1.31 [2 t, 3H þ 3H, N(CH2CH3)2],
2.03 [m, 1H, NCH2CH(CH3)2], 2.70–2.93 [m, 1H, 1H of NCH2CH(CH3)2],
2.83 (s, 3H, 9-CH3), 3.15–3.46 [m, 4H, 3H of N(CH2CH3)2 þ 1H of
NCH2CH(CH3)2], 4.16 [m, 1H, 1H of N(CH2CH3)2], 5.30f (near d, 1H, NH),
6.97 (dd, 1H, H-3), 7.03 (s, 1H, H-8), 8.17 (dd, 1H, H-4), 8.39 (dd, 1H,
H-2).
2j
70
70
157–157.5 (D)
144–145 (F)
C21H28N6O
1631 s (CO), 1601,
1581, 1541, 1501 w.
1.16 and 1.37 [2 t, 3H þ 3H, N(CH2CH3)2], 2.32–3.52 [m, 11H, piperazine
CH2’s þ 3H of N(CH2CH3)2], 2.39 (s, 3H, NCH3), 2.96 (near s, 3H, 9-CH3),
4.09 [m, 1H, 1H of N(CH2CH3)2], 7.28 (near s, 1H, H-8), 7.42 (dd, 1H, H-3),
8.48 (dd, 1H, H-4), 8.65 (dd, 1H, H-2).
2k
C22H30N6O
1637 s (CO), 1602,
1581, 1542, 1505 w.
1.12 (t, 3H, NCH2CH3), 1.13 and 1.34 [2 t, 3H þ 3H, N(CH2CH3)2], 2.29–
3.58 [m, 11H, piperazine CH2’s þ 3H of N(CH2CH3)2], 2.50 (q, 2H,
NCH2CH3), 2.93 (near s, 3H, 9-CH3), 4.03 [m, 1H, 1H of N(CH2CH3)2], 7.25
(near s, 1H, H-8), 7.38 (dd, 1H, H-3), 8.44 (dd, 1H, H-4), 8.62 (dd, 1H,
H-2).
2l
64
55
68
35
192–193 (B)
151–153 (A)
173–174 (C)
173–175 (A)
C27H32N6O
C22H30N6O2
C22H31N7O
C23H30N6O3
1635 s (CO), 1602,
1579, 1542.
1.14 and 1.33 [2 t, 3H þ 3H, N(CH2CH3)2], 2.29–3.51 [m, 11H, piperazine
CH2’s þ 3H of N(CH2CH3)2], 2.93 (s, 3H, 9-CH3), 3.60 (s, 2H, NCH2C6H5),
4.09 [m, 1H, 1H of N(CH2CH3)2], 7.18–7.47 (m, 6H, H-3 þ C6H5), 7.35 (s,
1H, H-8), 8.47 (dd, 1H, H-4), 8.61 (dd, 1H, H-2).
2m
2n
1628 s (CO), 1600,
1580, 1563, 1541 w,
1505 w.
1.11 and 1.32 [2 t, 3H þ 3H, N(CH2CH3)2], 1.52 (t, 3H, OCH2CH3), 2.17–
3.55 [m, 11H, piperazine CH2’s þ 3H of N(CH2CH3)2], 2.35 (s, 3H, NCH3),
4.01 [m, 1H, 1H of N(CH2CH3)2], 4.29 (m, 2H, OCH2CH3), 6.92 (s, 1H, H-8),
7.38 (dd, 1H, H-3), 8.40 (dd, 1H, H-4), 8.67 (dd, 1H, H-2).
1630 s (CO), 1599,
1579, 1556, 1538 w,
1509 w.
1.11 and 1.31 [2 t, 3H þ 3H, N(CH2CH3)2], 2.22–3.52 [m, 11H, piperazine
CH2’s þ 3H of N(CH2CH3)2], 2.34 (s, 3H, NCH3), 2.90 [s, 6H, N(CH3)2], 4.01
[m, 1H, 1H of N(CH2CH3)2], 7.03 (s, 1H, H-8), 7.37 (dd, 1H, H-3), 8.41 (dd,
1H, H-4), 8.71 (dd, 1H, H-2).
2o
1738 s (ester CO),
1636 s (amide CO),
1603, 1581, 1548,
1515 w.
1.17 and 1.32 [2 t, 3H þ 3H, N(CH2CH3)2], 1.37 (t, 3H, COOCH2CH3), 2.25–
3.58 [m, 11H, piperazine CH2’s þ 3H of N(CH2CH3)2], 2.40 (s, 3H, NCH3),
3.99 [m, 1H, 1H of N(CH2CH3)2], 4.38 (m, 2H, COOCH2CH3), 7.48 (dd, 1H,
H-3), 8.45 (dd, 1H, H-4), 8.67 (dd, 1H, H-2), 8.89 (s, 1H, H-9).
a
For the substituents, see Table 2.
b
Crystallization solvent: A ¼ diisopropyl ether, B ¼ ethyl acetate, C ¼ ethyl acetate/petroleum ether, D ¼ ethyl acetate/diisopropyl ether, E ¼ acetone/ethyl acetate,
F ¼ diisopropyl ether/petroleum ether.
c
Anal. C,H,N.
d
In KBr pellets. Abbreviations: s ¼ strong, w ¼ weak, br ¼ broad.
e
In CDCl3 solutions. Abbreviations: s ¼ singlet, br s ¼ broad singlet, d ¼ doublet, dd ¼ double doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet. J values for H-2, H-3, H-4 signals
(dd) of all naphthyridine derivatives: J2,3 ¼ J3,2 ¼ 4.7 Hz, J2,4 ¼ J4,2 ¼ 1.7 Hz, J3,4 ¼ J4,3 ¼ 8.1 Hz. J values for H-8 and H-9 signals (d) of compounds 2a–g: J8,9 ¼ J9,8 ¼ 1.4 Hz.
f
Disappeared with D2O.
activity (Table 2), and the structure of 1g, the novel most potent
anti-inflammatory compound 1, derives from the replacement
of the 6-CON(C2H5)2 substituent of the reference compound 1b,
with the 6-CONHC2H5 group. However, the replacement, in the
very potent anti-inflammatory lead compound 1a [4] (Table 1),
of the 6-CON(C2H5)2 substituent with the 6-CON(CH3)C2H5 one
afforded the compound 1h (Table 2) which showed, only at the
100 mg kgꢀ1 dose, significant anti-inflammatory activity
(inhibition 46%, P < 0.05) and analgesic activity (inhibition 82%,
P < 0.01), but no activities at the 50 mg kgꢀ1 dose. These latter
examples clearly indicate that an even little modification of
a significant substituent can maintain or increase (compound
1g), but also notably reduce (compound 1h) the activity of the
reference compound.
– As regards the substituted 5-aminoimidazo[1,2-a][1,8]naph-
thyridine-6-carboxamides 2a–o, their anti-inflammatory
activity is on the whole modest, whereas nearly all these
compounds, at the 100 mg kgꢀ1 dose, showed a statistically
significant antinociceptive activity and compounds 2b, c, j, k, m
proved to be notably active analgesic agents. It is interesting to
Only three compounds exhibited a low statistically significant
anti-inflammatory activity: compound 2a at the 100 mg kgꢀ1
(inhibition 29%, P < 0.05) and down to the 25 mg kgꢀ1 dose (inhi-
bition 25%, P < 0.05), and compounds 2f, h only at the 100 mg kgꢀ1
dose (inhibitions: 26% P < 0.05, and 38% P < 0.01, respectively).
4. Conclusions
Taking into account the pharmacological data of the new
compounds 1f–k and 2a-o described in the present paper (Table 2),
the following conclusions can be drawn.
– The above and other similar results previously obtained (Table
1) suggest that the tricyclic system of the 5-amino[1,2,4]-
triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 1
is an effective scaffold to obtain potent analgesic and/or anti-
inflammatory agents, depending on their substituents.
– For instance, among the new compounds 1f–k herein
described, 1g, i, j showed notably potent anti-inflammatory

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
361
(KBr): 3200–2500 br (NH), 1644 s, br (CO),1598, 1562 w, 1503 cmꢀ1
.
observe that the 5-amino substituent of the most potent
analgesic compounds 1 (1e) (Table 1) and 2 (2j, m) (Table 2) is
the same, i.e. 4-methyl-1-piperazinyl, most likely also due to
their isosteric tricyclic systems.
Anal. (C12H12ClN3O2) C, H, N.
5.1.2. 2,4-Dichloro-N-ethyl-N-methyl-1,8-naphthyridine-3-
carboxamide (4a)
– As above reported, compounds 1f–j (effective as anti-inflam-
matory agents) and 1k, 2a–h, j, k, m–o (antinoci-
ceptive agents) proved to be completely devoid of acute gas-
trolesivity in rats. These results are consistent with the data
obtained for compound 1b whose anti-inflammatory activity
was previously shown to derive from its ability to counteract
polymorphonuclear cells adhesion to endothelial cells and the
inhibition of both superoxide anion production and myelo-
peroxidase release, without involvement of COX inhibition [5].
– The mechanism underlying the antinociceptive activity of the
new compounds 2 remains to be defined as well as for the
previously studied compound 1e, whose very potent analgesic
activity (Table 1) was proved to be not related to the involvement
A mixture of 2.0 g of 3 (7.53 mmol) and 20 mL of POCl3 was
stirred at 110 ꢁC for 45 min. The excess POCl3 was removed by
heating at reduced pressure and the residue was dissolved in warm
water; the resulting solution was cooled, carefully treated with
NaHCO3 up to pH 7, and finally was exhaustively extracted with
dichloromethane. The combined extracts (dried over anhydrous
Na2SO4 and evaporated to dryness at reduced pressure) afforded
a thick oil which was chromatographed on a silica gel column,
eluting with dichloromethane-ethyl acetate (1:1). From the eluate
collected, after removal of solvents, a thick oil was obtained from
which, after adding a little diisopropyl ether, pure compound 4a
separated out (1.88 g, 88%) as white needles, m.p. 117–118 ꢁC, after
crystallization from diisopropyl ether. 1H NMR (CDCl3)
d 1.18 and
of m-opioid, muscarinic, nicotinic and serotonin receptors [4].
1.30 (2 t, 3H, CONCH2CH3), 2.89 and 3.19 (2s, 3H, CONCH3), 3.24 and
3.69 (q þ m, 2H, CONCH2CH3), 7.65 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼4.4 Hz,
1H, H-6), 8.58 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5), 9.17 (dd,
J7,6 ¼ 4.4 Hz, J7,5 ¼1.8 Hz, 1H, H-7); IR (KBr): 1640 s (CO), 1594, 1578,
1547 cmꢀ1. Anal. (C12H11Cl2N3O) C, H, N.
5. Experimental protocols
5.1. Chemistry
Melting points were determined using a Fisher-Johns apparatus
and are uncorrected. IR spectra were recorded on a Perkin–Elmer
‘‘Spectrum One’’ spectrophotometer (abbreviations relative to IR
bands: br ¼ broad, s ¼ strong, w ¼ weak, sh ¼ shoulder). 1H NMR
spectra were recorded on a Varian Gemini 200 (200 MHz) spec-
5.1.3. (2,4-Dichloro-1,8-naphthyridin-3-yl)-(4-methyl-1-
piperazinyl)-methanone (4b)
A mixture of 25.0 mmol (5.50 g) of ester 6 [7], 50 mL of ethanol
and an excess (10 mL) of 1-methylpiperazine was heated in a closed
vessel at 160 ꢁC for 16 h. After cooling, the resulting suspension was
transferred into a flask and evaporated to dryness at reduced
pressure. To the resulting resinous residue (crude compound 7)
POCl3 (50 mL) was added and the mixture was stirred at 110 ꢁC for
2 h. Starting from this point, the reaction work-up was identical to
that above described for preparation of 4a, except for the elution of
silica gel column which was performed using a mixture dichloro-
methane–petroleum ether–triethylamine (6:2:1). From the eluate
collected, after removal of solvents, a nearly solid residue was
obtained from which, after adding a little diisopropyl ether, pure 4b
separated out (3.33 g, 41% referring to 6); whitish crystals, m.p.
186–187 ꢁC, after crystallization from ethyl acetate/petroleum
trometer, using (CH3)4Si as an internal reference (
d
¼ 0), and
chemical shifts are reported in ppm. Spin multiplicities are given as
follows: s (singlet), br s (broad singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), dd (double doublet). Analyses of all new
compounds, indicated by the symbols of the elements, were within
ꢂ0.4% of the theoretical values and were performed by the Labo-
ratorio di Microanalisi, Dipartimento di Scienze Farmaceutiche,
`
Universita di Genova. Thin layer chromatograms were run on Merck
silica gel 60 F254 precoated plastic sheets (layer thickness 0.2 mm).
Column chromatography was performed using Carlo Erba silica gel
(0.05–0.20 mm) or Carlo Erba neutral aluminium oxide (Brock-
mann activity I).
ether with charcoal. 1H NMR (CDCl3):
d 2.33 (s, 3H, NCH3), 2.39
(near q, 2H, 2H of piperazine), 2.55 (near t, 2H, 2H of piperazine),
3.30 (near q, 2H, 2H of piperazine), 3.91 (m, 2H, 2H of piperazine),
7.66 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼4.4 Hz, 1H, H-6), 8.59 (dd, J5,6 ¼ 8.4 Hz,
J5,7 ¼ 1.8 Hz, 1H, H-5), 9.19 (dd, J7,6 ¼ 4.4 Hz, J7,5 ¼1.8 Hz, 1H, H-7);
IR (KBr): 1645 s (CO), 1596, 1582, 1543 cmꢀ1. Anal. (C14H14Cl2N4O)
C, H, N.
5.1.1. 4-Chloro-1,2-dihydro-N-ethyl-N-methyl-2-oxo-1,8-
naphthyridine-3-carboxamide (3)
Phosphorus oxychloride (75.0 mmol, 11.50 g) was added drop-
wise to an ice-cooled mixture of 2-aminonicotinic acid (37.5 mmol,
5.18 g) and ethyl N-ethyl-N-methylmalonamate [6] (75.0 mmol,
12.99 g) and the resulting slurry was heated at 110 ꢁC for 3 h. The
dark and sticky mixture so obtained was dissolved in hot water
(200 mL) and treated with CH3COONa$3H2O (500 mmol, 68 g),
stirring then the mixture at room temperature for 1 h. The subse-
quent exhaustive extraction with dichloromethane, followed by
evaporation to dryness under reduced pressure of the combined
extracts (dried over anhydrous Na2SO4), afforded a thick oil which
was chromatographed on a silica gel column eluting with the
mixture dichloromethane-ethyl acetate (1:1). The fractions
collected, after removal of solvents, gave a pasty solid residue,
which was taken up in a little ethyl acetate and filtered to give the
nearly pure compound 3 (2.29 g, 23%); white needles, m.p. 225–
226 ꢁC, after crystallization from ethyl acetate with charcoal. 1H
5.1.4. General procedure for the synthesis of compounds 5a, b
A mixture of 6.0 mmol of 4a (1.70 g) or 4b (1.95 g), 9.0 mmol
(0.92 g) of isobutyrohydrazide and 10 mL of Dowtherm A was stir-
red at 160 ꢁC for 30 min. After cooling, 10% aqueous Na2CO3 (50 mL)
and dichloromethane (50 mL) were added and the mixture was
further stirred at room temperature for 30 min. After discarding
some insoluble impurities by filtration, the mixture was transferred
in a separatory funnel, then the organic layer was collected and the
aqueous one was exhaustively extracted with dichloromethane.
The combined organic phases were dried (anhydrous Na2SO4) then
evaporated to dryness at reduced pressure, and the residue was
subjected to chromatography on a silica gel column, eluting first
with dichloromethane to remove Dowtherm A. The reaction
products were then recovered eluting with the mixture ethyl
acetate–tetrahydrofuran (4:1) (compound 5a) or dichloromethane–
petroleum ether–triethylamine (6:2:1) (compound 5b). The eluate
NMR (CDCl3):
d 1.20 and 1.28 (2 t, 3H, CONCH2CH3), 2.97 and 3.15
(2s, 3H, CONCH3) 3.31, 3.52 and 3.80 (q þ 2 m, 2H, CONCH2CH3),
7.34 (dd, J6,5 ¼ 8 Hz, J6,7 ¼4.8 Hz, 1H, H-6), 8.32 (dd, J5,6 ¼ 8 Hz,
J5,7 ¼ 1.6 Hz, 1H, H-5), 8.86 (dd, J7,6 ¼ 4.8 Hz, J7,5 ¼1.6 Hz, 1H, H-7),
12.20–13.40 (broad signal, 1H, NH; disappeared with D2O); IR

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G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
collected, after removal of solvents, gave an oily residue from which
treatment with a little ethyl acetate and diethyl ether, afforded pure
compounds 5a, b were obtained as described below.
compound 1i as whitish crystals.
5.1.5.5. N,N-Diethyl-9-isopropyl-5-(propylamino)[1,2,4]triazolo
[4,3-a][1,8]naphthyridine-6-carboxamide (1l). The oily residue
obtained from reaction of 5d [3] (1.38 g) with propylamine, after
treatment with a little diisopropyl ether, afforded 1.34 g (91%) of 1l;
whitish crystals, m.p. 160–162 ꢁC, after crystallization from ethyl
5.1.4.1. 5-Chloro-N-ethyl-N-methyl-9-isopropyl[1,2,4]triazolo[4,3-a]-
[1,8]naphthyridine-6-carboxamide (5a). The residue obtained from
the reaction performed with 4a was treated with a little diethyl
ether/petroleum ether so that the nearly pure 5a separated out as
a whitish crystalline solid (0.80 g, 40%); white crystals, m.p. 150 ꢁC,
after crystallization from ethyl acetate/petroleum ether. 1H NMR
acetate/petroleum ether. 1H NMR (CDCl3):
d 1.01 (t, 3H,
NCH2CH2CH3),1.16 and 1.32 [2 t, 3H þ 3H, N(CH2CH3)2],1.34 and 1.61
[2d, 3H þ 3H, 9-CH(CH3)2], 1.80 (m, 2H, NCH2CH2CH3), 2.93 [m, 1H,
1H of NCH2CH2CH3], 3.27–3.63 [m, 4H, 3H of N(CH2CH3)2 þ 1H of
NCH2CH2CH3], 3.82 [m, 1H, 1H of N(CH2CH3)2], 4.12 [m, 1H, 9-
CH(CH3)2], 6.36 (near d, 1H, NH; disappeared with D2O), 6.92 (dd,
J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.7 Hz,1H, H-3), 8.18 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.7 Hz,
1H, H-4), 8.28 (dd, J2,3 ¼ 4.7 Hz, J2,4 ¼1.7 Hz,1H, H-2); IR (KBr): 3263
(NH), 1614 s, br (CO), 1545, 1518 w cmꢀ1. Anal. (C20H28N6O) C, H, N.
(CDCl3):
d 1.19 and 1.34 (2t, 3H, CONCH2CH3), 1.52 and 1.59 [2d,
3H þ 3H, 9-CH(CH3)2], 2.96 and 3.23 (2s, 3H, CONCH3) 3.31 and
3.55–3.94 (q þ m, 2H, CONCH2CH3), 4.50 [m, 1H, 9-CH(CH3)2], 7.61
(dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.7 Hz, 1H, H-3), 8.54 (dd, J4,3 ¼ 8.1 Hz,
J4,2 ¼ 1.7 Hz, 1H, H-4), 8.77 (dd, J2,3 ¼ 4.7 Hz, J2,4 ¼1.7 Hz, 1H, H-2);
IR (KBr): 1650 s (CO), 1602, 1589, 1559, 1519 w cmꢀ1. Anal.
(C16H18ClN5O) C, H, N.
5.1.4.2. (5-Chloro-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]naphthyridin-6-
yl)-(4-methyl-1-piperazinyl)-methanone (5b). The oily residue
derived from the reaction carried out with 4b, treated with a little
diisopropyl ether, afforded pure 5b as white solid (0.68 g, 30%); m.p.
220–221 ꢁC after crystallization from ethyl acetate/petroleum ether.
5.1.6. N,N-Diethyl-9-ethyl-5-(N-isobutyl,N-methylamino)[1,2,4]
triazolo[4,3-a][1,8]naphthyridine-6-carboxamide (1f) and N,N-
diethyl-9-isopropyl-5-(N-methyl,N-propylamino)[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide (1j)
To the suspension of 3.0 mmol (1.10 g) of 1m [1] or 1l in 80 mL of
2-butanone, 24 mmol (1.34 g) of finely powdered KOH mixed with
2.0 g of anhydrous K2CO3 were added, then the mixture was
refluxed (80 ꢁC) for 5 min. A solution of 4.0 mmol (0.25 mL) of
iodomethane in 10 mL of 2-butanone was then slowly added and
the resulting mixture was further refluxed for 1.5 h, with stirring.
After cooling, the solvent was removed in vacuo and the residue
was partitioned between water (200 mL) and dichloromethane
(200 mL). The organic layer was collected and the aqueous one was
further extracted twice with dichloromethane. The combined
organic phases were dried (anhydrous Na2SO4), then evaporated to
dryness in vacuo to give an oil which was chromatographed on
a silica gel column eluting first with ethyl acetate. The first fractions
of this eluate, containing some impurities, were discarded.
Compounds 1f, j were then recovered eluting with the mixture
ethyl acetate-acetone (2:1). The eluate collected, evaporated to
dryness in vacuo, afforded an oil from which, after treatment with
a little diisopropyl ether, pure compounds 1f, j separated out as pale
yellow solids.
1H NMR (CDCl3):
d
1.53 and 1.58 [2d, 3H þ 3H, 9-CH(CH3)2], 2.65 (s,
3H, NCH3), 2.72–3.29, 3.47–3.63 and 3.76–4.00 (3 m, 4H þ 2H þ 2H,
piperazine CH2’s), 4.49 [m, 1H, 9-CH(CH3)2], 7.65 (dd, J3,4 ¼ 8.1 Hz,
J3,2 ¼ 4.7 Hz, 1H, H-3), 8.57 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.7 Hz, 1H, H-4),
8.81 (dd, J2,3 ¼ 4.7 Hz, J2,4 ¼1.7 Hz, 1H, H-2); IR (KBr): 1650 s (CO),
1600, 1588, 1558, 1517 w cmꢀ1. Anal. (C18H21ClN6O) C, H, N.
5.1.5. Synthesis of the 5-amino-9-isopropyl[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide derivatives 1g, h, i, k, l
A mixture of the proper chloroderivative 5 (4.0 mmol) and the
proper amine (5 mL) in 25 mL of anhydrous ethanol was heated at
140 ꢁC in a closed vessel for 16 h. After cooling, the resulting solution
was evaporated to dryness at reduced pressure and the residue was
partitioned between 10% aqueous Na2CO3 (100 mL) and dichloro-
methane (100 mL). The organic layer was collected and the aqueous
one was further extracted twice with dichloromethane. The
combined organic phases were dried (anhydrous Na2SO4), then
evaporated to dryness at reduced pressure to give an oily or solid
residue, from which, after treatment with suitable solvents, pure
compounds 1g, h, i, k, l separated out as crystalline solids. According
to this procedure the following compounds were obtained:
Data for compounds 1f–k are reported in Table 3.
5.1.7. 4-Chloro-2-[(2,2-dimethoxyethyl)amino]-N,N-diethyl-1,8-
naphthyridine-3-carboxamide (8a) and 2-chloro-4-[(2,2-
5.1.5.1. N-ethyl-N-methyl-9-isopropyl-5-(isopropylamino)[1,2,4]tri-
azolo[4,3-a][1,8]naphthyridine-6-carboxamide (1h). The thick oil
obtained from reaction of 5a (1.33 g) with isopropylamine, after
treatment with a little diethyl ether, afforded pure 1h as pale yellow
crystals.
dimethoxyethyl)amino]-N,N-diethyl-1,8-naphthyridine-3-
carboxamide (9a)
A mixture of the 2,4-dichloroderivative 4c [8] (5.0 mmol,1.49 g),
(2,2-dimethoxyethyl)amine (50.0 mmol, 5.26 g), and anhydrous
ethanol (50 mL) was stirred at room temperature for 2 h. The
resulting solution was poured into water (250 mL) and the mixture
was exhaustively extracted with dichloromethane. The combined
extracts (dried over anhydrous Na2SO4), was evaporated to dryness
under reduced pressure to give a thick oil which was chromato-
graphed on a silica gel column, eluting with the mixture petroleum
ether-dichloromethane-triethylamine (6:3:1) to recover compound
8a. The fraction collected, after removal of solvents, gave compound
8a as a resinous oil (0.81 g, 44%). In order to obtain an analytical
sample, a little amount of 8a was subjected to a second column
chromatography [silica gel, ethyl acetate–tetrahydrofuran (4:1) as
eluant] to give pure 8a as a whitish resinous oil. 1H NMR (CDCl3):
5.1.5.2. [5-(Isobutylamino)-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]na-
phthyridin-6-yl]-(4-methyl-1-piperazinyl)-methanone
(1k). The
solid obtained from reaction of 5b (1.49 g) with isobutylamine was
taken up in a little diethyl ether and filtered to give pure 1k as
whitish crystals.
5.1.5.3. N-Ethyl-5-(isobutylamino)-9-isopropyl[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide (1g). The thick oil obtained
from reaction of 5c [4] (1.27 g) with isobutylamine, after treatment
with a little diisopropyl ether, afforded pure 1h as yellow crystals.
d
1.06 and 1.26 [2 t, 3H þ 3H, N(CH2CH3)2], 2.95–3.95 [m, 6H,
5.1.5.4. 5-(Allylamino)-N,N-diethyl-9-isopropyl[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide (1i). The resinous residue
obtained from reaction of 5d [3] (1.38 g) with allylamine, after
N(CH2CH3)2 þ NCH2CH(OCH3)2], 3.34 and 3.37 [2s, 3H þ 3H,
NCH2CH(OCH3)2], 4.51 [t,1H, NCH2CH(OCH3)2], 5.27 (near t,1H, NH;
disappeared with D2O), 7.21 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼4.4 Hz, 1H, H-6),

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
363
8.28 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5), 8.82 (dd, J7,6 ¼ 4.4 Hz,
J7,5 ¼1.8 Hz, 1H, H-7); IR (CHCl3): 3432 (NH), 1628 s (CO), 1610 s,
1557, 1530 cmꢀ1. Anal. (C17H23ClN4O3) C, H, N. As a crystalline
derivative of 8a, its picrate was prepared: yellow crystals, m.p. 178–
180 ꢁC, after crystallization from ethanol. Anal. (C23H26ClN7O10) C,
H, N.
at reduced pressure to give an oily residue which was subjected to
chromatography on a silica gel column eluting first with dichloro-
methane to remove Dowtherm A. The pure compounds 10a or 10b
were then recovered eluting with the mixture ethyl acetate–
tetrahydrofuran (4:1) and crystallized from the proper solvent. In
this way were obtained:
The elution was then pursued with tetrahydrofuran: the eluate
collected, evaporated to dryness in vacuo, afforded a thick oil from
which, after treatment with a little diethyl ether, pure compound
9a separated out as a crystalline solid (0.75 g, 41%); white crystals,
m.p. 139–140 ꢁC, after crystallization from ethyl acetate/diisopropyl
5.1.9.1. 5-Chloro-N,N-diethylimidazo[1,2-a][1,8]naphthyridine-6-car-
boxamide (10a). Obtained from 8a (1.29 g, 85%); white crystals,
m.p. 188–190 ꢁC, after crystallization from diisopropyl ether. 1H
NMR (CDCl3):
d
1.14 and 1.39 [2 t, 3H þ 3H, N(CH2CH3)2], 3.29 [q, 2H,
ether. 1H NMR (CDCl3):
d
1.11 and 1.27 [2 t, 3H þ 3H, N(CH2CH3)2],
2H of N(CH2CH3)2], 3.50 and 3.99 [2 m, 1H þ 1H, 2H of N(CH2CH3)2],
7.59 (dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.7 Hz,1H, H-3), 7.68 (d, J8,9 ¼ 1.4 Hz,1H,
H-8), 8.46 (d, J9,8 ¼ 1.4 Hz, 1H, H-9), 8.58 (dd, J4,3 ¼ 8.1 Hz,
J4,2 ¼ 1.7 Hz, 1H, H-4), 8.78 (dd, J2,3 ¼ 4.7 Hz, J2,4 ¼1.7 Hz, 1H, H-2);
IR (KBr): 1633 s (CO), 1595 w, 1584, 1550, 1520 cmꢀ1. Anal.
(C15H15ClN4O) C, H, N.
3.17–3.83 [m, 6H, N(CH2CH3)2 þ NCH2CH(OCH3)2], 3.39 and 3.42
[2s, 3H þ 3H, NCH2CH(OCH3)2], 4.54 [t, 1H, NCH2CH(OCH3)2], 5.53
(near t, 1H, NH; disappeared with D2O), 7.37 (dd, J6,5 ¼ 8.4 Hz,
J6,7 ¼4.4 Hz, 1H, H-6), 8.17 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5),
8.96 (dd, J7,6 ¼ 4.4 Hz, J7,5 ¼1.8 Hz, 1H, H-7); IR (KBr): 3328 (NH),
1620 s (CO), 1597, 1564 s, br cmꢀ1. Anal. (C17H23ClN4O3) C, H, N.
5.1.9.2. 5-Chloro-N,N-diethyl-9-methylimidazo[1,2-a][1,8]naphthyr-
idine-6-carboxamide (10b). Obtained from 8b (1.27 g, 80%); white
crystals, m.p. 200.5–201 ꢁC, after crystallization from acetone. 1H
5.1.8. 4-Chloro-N,N-diethyl-2-(propargylamino)-1,8-
naphthyridine-3-carboxamide (8b) and 2-chloro-N,N-diethyl-4-
(propargylamino)-1,8-naphthyridine-3-carboxamide (9b)
NMR (CDCl3):
d
1.15 and 1.40 [2 t, 3H þ 3H, N(CH2CH3)2], 3.01 (s, 3H,
A mixture of the 2,4-dichloroderivative 4c [8] (5.0 mmol, 1.49 g),
propargylamine (50.0 mmol, 2.75 g), and anhydrous ethanol
(50 mL) was stirred at room temperature for 24 h. Starting from this
point, the reaction work-up and the separation of isomers 8b, 9b by
column chromatography were identical to those above described
for preparation of isomers 8a, 9a. From the fraction eluted with the
mixture petroleum ether-dichloromethane-triethylamine (6:3:1)
pure 8b was obtained (0.71 g, 45%); white crystals, m.p. 177–178 ꢁC,
after crystallization from ethyl acetate/diisopropyl ether. 1H NMR
9-CH3), 3.30 [q, 2H, 2H of N(CH2CH3)2], 3.49 and 4.02 [2 m, 1H þ 1H,
2H of N(CH2CH3)2], 7.40 (s, 1H, H-8), 7.55 (dd, J3,4 ¼ 8.1 Hz,
J3,2 ¼ 4.7 Hz, 1H, H-3), 8.54 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.7 Hz, 1H, H-4),
8.75 (dd, J2,3 ¼ 4.7 Hz, J2,4 ¼1.7 Hz, 1H, H-2); IR (KBr): 1634 s (CO),
1604, 1584, 1540 cmꢀ1. Anal. (C16H17ClN4O) C, H, N.
5.1.10. General procedure for the synthesis of 5-amino-N,N-
diethylimidazo[1,2-a][1,8]naphthyridine-6-carboxamide derivatives
2a, c–h, j–l
(CDCl3):
d
1.13 and 1.34 [2 t, 3H þ 3H, N(CH2CH3)2], 2.26 [t (prop-
A mixture of 4.0 mmol of 10a (1.21 g) or 10b (1.27 g) and an
excess (15 mL) of the proper 1-substituted piperazine (preparation
of compounds 2c–g, j–l) was heated (without solvent) in a closed
vessel at 160 ꢁC for 16 h. In the case of preparation of compounds 2a
or 2h were used 50 mL of a 2 M solution of ethylamine in methanol
or 25 mL of a 33% solution of methylamine in absolute ethanol,
respectively, and the mixture was heated in a closed vessel at
160 ꢁC for 16 h. After cooling, the desired compounds 2a, c–h, j–l
were obtained as reported below.
argylic coupling with NCH2–C^C–, J ¼ 2.4 Hz), 1H, NCH2–C^CH],
3.12–3.67 [m, 3H, 3H of N(CH2CH3)2], 3.90 [m, 1H, 1H of
N(CH2CH3)2], 4.33 and 4.59 [AB q (each peak of AB q is further
coupled with –C^CH and NH), J ¼ 20 Hz, 1H þ 1H, NCH2–C^CH],
5.32 (br s, 1H, NH; disappeared with D2O), 7.33 (dd, J6,5 ¼ 8.4 Hz,
J6,7 ¼4.4 Hz, 1H, H-6), 8.38 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5),
8.92 (dd, J7,6 ¼ 4.4 Hz, J7,5 ¼1.8 Hz, 1H, H-7); IR (KBr): 3300–3170 br
(C^CH þ NH), 2112 w (C^C), 1634 s (CO), 1605 s, 1532 s, br cmꢀ1
.
Anal. (C16H17ClN4O) C, H, N.
From the fraction eluted with tetrahydrofuran, pure 9b was
recovered (0.70 g, 44%); white crystals, m.p. 146–147 ꢁC, after
crystallization from ethyl acetate/petroleum ether. 1H NMR (CDCl3):
5.1.10.1. Compounds 2a, h (reactions of 10a with ethylamine or 10b
with methylamine, respectively). The final solution was poured
into water (150 mL) and the mixture was exhaustively extracted
with chloroform. The residue obtained from the combined extracts
(dried over anhydrous Na2SO4 and evaporated to dryness in vacuo)
was chromatographed on a aluminium oxide column, eluting with
ethyl acetate to remove small amounts of starting compounds 10a
or 10b, respectively. The subsequent elution with tetrahydrofuran
afforded pure compound 2a or 2h (pale yellow solids), which were
crystallized from the proper solvents.
d
1.14 and 1.29 [2 t, 3H þ 3H, N(CH2CH3)2], 2.35 [t (propargylic
coupling with NCH2–C^C–, J ¼ 2.4 Hz), 1H, NCH2–C^CH], 3.27 [q,
2H, 2H of N(CH2CH3)2], 3.47 and 3.75 [2 m, 1H þ 1H, 2H of
N(CH2CH3)2], 4.20 and 4.33 [AB q (each peak of AB q is further
coupled with –C^CH and NH), J ¼ 20 Hz, 1H þ 1H, NCH2–C^CH],
5.79 (near t, 1H, NH; disappeared with D2O), 7.38 (dd, J6,5 ¼ 8.4 Hz,
J6,7 ¼4.4 Hz, 1H, H-6), 8.37 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5),
8.96 (dd, J7,6 ¼ 4.4 Hz, J7,5 ¼1.8 Hz, 1H, H-7); IR (KBr): 3320 sh
(C^CH), 3286 (NH), 2117 w (C^C),1634 s (CO),1600,1570 s,1559 s,
1539 s, cmꢀ1. Anal. (C16H17ClN4O) C, H, N.
5.1.10.2. Compounds 2c, d, j, k (reactions of 10a or 10b with 1-
methyl- or 1-ethylpiperazine). The excess of amine was removed
in vacuo and the residue was partitioned between dichloro-
methane (100 mL) and 10% aqueous Na2CO3 (50 mL). The aqueous
phase was further extracted twice with dichloromethane. The
residue obtained from the combined extracts (dried over anhy-
drous Na2SO4 and evaporated to dryness in vacuo) was chroma-
tographed on a silica gel column, eluting with ethyl acetate to
remove small amounts of starting compounds 10a or 10b and
impurities. The subsequent elution with the mixture dichloro-
methane-triethylamine (9:1) afforded pure compounds 2c, d, j, k
(as pale yellow solids) which were then crystallized from the
proper solvents.
5.1.9. General method for the synthesis of compounds 10a, b
A mixture of 8a (5.0 mmol, 1.83 g) or 8b (5.0 mmol, 1.58 g),
monohydrate p-toluenesulphonic acid (5.0 mmol, 0.95 g) and
Dowtherm A (10 mL) was stirred at 180 ꢁC for 20 min. After cooling,
10% aqueous Na2CO3 (50 mL) and dichloromethane (50 mL) were
added and the mixture was further stirred at room temperature for
30 min. The mixture was transferred in a separatory funnel, then
the organic layer was collected and the aqueous one was exhaus-
tively extracted with dichloromethane. The combined organic
phases were dried (anhydrous Na2SO4) then evaporated to dryness

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G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
5.1.10.3. Compounds 2f, g, l. Reactions of 10a or 10b with 1-benzyl-
or 1-(2-methoxyphenyl)piperazine. The viscous liquid obtained
was dissolved in ethanol and the solution poured into abundant
water (400 mL) so that the nearly pure compounds 2f, g, l separated
out as yellow solids that were recovered by filtration, washed with
water, dried, then crystallized from the proper solvents.
(3.0 mmol, 0.57 g) and Dowtherm A (10 mL) was stirred at 180 ꢁC
for 20 min. By proceeding exactly as above described for isolation of
2b, the pure compound 2i was finally obtained as yellow crystalline
solid whose data are reported in Table 3.
5.1.14. Ethyl [[3-(diethylcarbamoyl)-4-(4-methyl-1-piperazinyl)-
1,8-naphthyridin-2-yl]amino]acetate (11b)
5.1.10.4. Compound 2e. The viscous liquid derived from reaction of
10a with 1-(2-hydroxyethyl)piperazine was poured into abundant
water and the resulting emulsion was exhaustively extracted with
chloroform. The combined extracts (dried over anhydrous Na2SO4
and evaporated to dryness in vacuo) afforded a thick oil from which,
after treatment with a little diethyl ether, the pure 2e separated out
as yellow crystalline solid, which was crystallized from the proper
solvent.
A mixture of the glycine ethyl ester derivative 8c [9] (4.0 mmol,
1.46 g), 1-methylpiperazine (5.0 mL) and dimethyl sulphoxide
(5.0 mL) was heated at 130 ꢁC for 3 h with stirring. The resulting
solution was poured into water (250 mL) and the mixture was
exhaustively extracted with chloroform. The combined extracts,
dried over anhydrous Na2SO4 and evaporated to dryness in vacuo,
afforded a thick oil which was chromatographed on a silica gel
column eluting first with the mixture ethyl acetate–tetrahydro-
furan (4:1) to remove some impurities, then with the mixture
dichloromethane-triethylamine (9:1) to recover 11b. The eluate
collected, after removal of solvents, gave pure 11b (1.34 g, 78%), as
whitish resinous oil. A share of this oil (0.64 g, 1.5 mmol) was
treated, at room temperature, with a solution of maleic acid
(3.0 mmol, 0.35 g) in anhydrous ethanol to give the corresponding
dimaleate (11b$2H4C4O4) as a white solid, m.p. 174–175 ꢁC, after
crystallization from anhydrous ethanol/diethyl ether. Anal.
(C22H32N6O3$2H4C4O4) C, H, N. The 1H NMR and IR spectra were
recorded on the free amine obtained (thick oil) from the dimaleate
by treatment with aqueous sodium bicarbonate, exhaustive
extraction with chloroform and removal of solvent. 1H NMR
Data for compounds 2a, c–h, j–l are reported in Table 3.
5.1.11. N,N-Diethyl-2-[(2,2-dimethoxyethyl)amino]-4-
(isobutylamino)-1,8-naphthyridine-3-carboxamide (11a)
A
mixture of 2-chloro-N,N-diethyl-4-(isobutylamino)-1,8-
naphthyridine-3-carboxamide 9c [9] (4.0 mmol, 1.34 g), (2,2-
dimethoxyethyl)amine (40.0 mmol, 4.20 g), and ethylene glycol
(10 mL) was stirred at 140 ꢁC for 2 h. The resulting solution was
poured into water (250 mL) and the mixture was exhaustively
extracted with dichloromethane. The combined extracts (dried
over anhydrous Na2SO4), was evaporated to dryness under reduced
pressure to give a thick oil which was chromatographed on a silica
gel column, eluting with the mixture acetone-ethyl acetate (1:1).
The eluate collected, evaporated to dryness in vacuo, gave an oily
residue from which, after addition of a little diisopropyl ether, pure
compound 11a separated out as a whitish solid (1.18 g, 73%); white
crystals, m.p. 144–145 ꢁC, after crystallization from ethyl acetate/
(CDCl3):
d
1.10 and 1.35 [2 t, 3H þ 3H, N(CH2CH3)2], 1.26 (t, 3H,
COOCH2CH3), 2.23–2.76 and 3.01–3.48 [2 m, 10H, piperazine
CH2’s þ 2H of N(CH2CH3)2], 2.40 (s, 3H, NCH3), 3.62 [q, 2H, 2H of
N(CH2CH3)2], 4.19 (q, 2H, COOCH2CH3), 4.20 and 4.56 [AB q,
J ¼ 18 Hz, 1H þ 1H, HNCH2COOC2H5; each peak of AB q is coupled
with NH (J ¼ 5.8 Hz): this coupling disappeared with D2O], 5.58
(near t, 1H, NH; disappeared with D2O), 7.11 (dd, J6,5 ¼ 8.4 Hz,
J6,7 ¼4.4 Hz, 1H, H-6), 8.15 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5),
8.74 (dd, J7,6 ¼ 4.4 Hz, J7,5 ¼1.8 Hz, 1H, H-7); IR (CHCl3): 3421 (NH),
diisopropyl ether. 1H NMR (CDCl3):
d 0.90–1.37 [m, 6H,
N(CH2CH3)2], 0.98 [d, 6H, NCH2CH(CH3)2], 1.90 [m, 1H,
NCH2CH(CH3)2], 2.97–3.67 [m, 6H, N(CH2CH3)2 þ NCH2CH(CH3)2],
3.40 [near s, 6H, NCH2CH(OCH3)2], 3.79 [m (d after treatment with
D2O), 2H, NCH2CH(OCH3)2], 4.56 [t, 1H, NCH2CH(OCH3)2], 4.73 (near
t, 1H, NH; disappeared with D2O), 5.41 (near t, 1H, NH; disappeared
with D2O), 7.10 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼4.4 Hz, 1H, H-6), 8.01 (dd,
J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5), 8.75 (dd, J7,6 ¼ 4.4 Hz,
J7,5 ¼1.8 Hz, 1H, H-7); IR (KBr): 3357 s (NH), 1607 s, br (CO), 1557 s,
1524 s cmꢀ1. Anal. (C21H33N5O3) C, H, N.
1736 (ester CO), 1618 s (amide CO), 1591 s, 1562, 1524 cmꢀ1
.
5.1.15. N,N-Diethyl-9-ethoxy-5-(4-methyl-1-piperazinyl)imidazo-
[1,2-a][1,8]naphthyridine-6-carboxamide (2m)
A mixture of 11b (5.0 mmol, 2.14 g), polyphosphoric acid (1.20 g)
and POCl3 (2.50 mL) was heated at 120 ꢁC for 3.5 h, with stirring.
The final resinous mixture was dissolved in hot water and the
resulting dark greenish solution was made alkaline with Na2CO3
and exhaustively extracted with dichloromethane. The combined
extracts, dried over anhydrous Na2SO4 and evaporated to dryness in
vacuo, afforded a thick oil which was chromatographed on a silica
gel column eluting with the mixture dichloromethane–petroleum
ether–triethylamine (6:3:1). The eluate collected gave an oily
residue from which, after addition of a little diisopropyl ether, pure
compound 2m separated out as a yellow crystalline solid whose
data are reported in Table 3.
5.1.12. N,N-Diethyl-5-(isobutylamino)imidazo[1,2-a]
[1,8]naphthyridine-6-carboxamide (2b)
A mixture of 11a (3.0 mmol, 1.21 g), monohydrate p-toluene-
sulphonic acid (3.0 mmol, 0.57 g) and Dowtherm A (10 mL) was
stirred at 180 ꢁC for 20 min. After cooling, 10% aqueous Na2CO3
(50 mL) and dichloromethane (50 mL) were added and the mixture
was further stirred at room temperature for 30 min. The mixture
was transferred in a separatory funnel, then the organic layer was
collected and the aqueous one was exhaustively extracted with
dichloromethane. The combined organic phases were dried
(anhydrous Na2SO4) then evaporated to dryness at reduced pres-
sure to give an oily residue which was subjected to chromatography
on a silica gel column eluting first with dichloromethane to remove
Dowtherm A. The subsequent elution with the mixture dichloro-
methane–petroleum ether–triethylamine (5:5:1) afforded the pure
compound 2b (as yellow crystalline solid) whose data are reported
in Table 3.
5.1.16. Ethyl 6-(diethylcarbamoyl)-5-(4-methyl-1-piperazinyl)
imidazo[1,2-a][1,8]naphthyridine-8-carboxylate (2o)
A
mixture of 11b (6.0 mmol, 2.57 g) and N,N-dime-
thylformamide diethyl acetal (5.0 mL) was heated at 120 ꢁC for
24 h, with stirring. The resulting dark solution was evaporated to
dryness in vacuo and the oily residue was dissolved in a little
dichloromethane and cromatographed on a silica gel column,
eluting with the mixture dichloromethane–petroleum ether–
triethylamine (5:5:1). The eluate collected gave an oily residue
from which, after addition of a little diisopropyl ether, pure
compound 2o separated out as a yellow crystalline solid whose
data are reported in Table 3.
5.1.13. N,N-Diethyl-5-(isobutylamino)-9-methylimidazo[1,2-a]
[1,8]naphthyridine-6-carboxamide (2i)
A
mixture of 9c [9] (3.0 mmol, 1.00 g), propargylamine
(30.0 mmol, 1.65 g), monohydrate p-toluenesulphonic acid

G. Roma et al. / European Journal of Medicinal Chemistry 45 (2010) 352–366
365
5.1.17. N,N-Dimethyl-[[3-(diethylcarbamoyl)-4-(4-methyl-1-
piperazinyl)-1,8-naphthyridin-2-yl]amino]acetamide (11c)
Comerio (VA), Italy). Edema values, obtained in the control group,
were considered arbitrarily as 100; the percentage of inhibition
was calculated from the difference in the swelling between the
treated and the control group.
To the solution of 11b (6.0 mmol, 2.57 g) in 10 mL of anhydrous
ethanol were added 10 mL of a 30% solution of dimethylamine in
absolute ethanol and the mixture was heated in a closed vessel at
150 ꢁC for 16 h. After cooling, the resulting solution was evaporated
to dryness in vacuo to give a thick oil which, after treatment with
a little diethyl ether, afforded pure compound 11c (2.00 g, 78%) as
a white crystalline solid, m.p. 193–195 ꢁC, after crystallization from
5.2.2. Analgesic activity
Antinociceptive activity was studied by writhing test [11],
through the intraperitoneal injection of 0.2 mL/mouse of acetic acid
solution (0.6%) (Sigma Aldrich, Milan, Italy) 1 h after oral treatment
with the test drugs. Complete extension of either hind limb was
regarded as a writhing response. The number of writhes of each
mouse was counted over a period of 30 min after the injection of
the noxious agent. The percentage of inhibition was calculated
from the difference in the writhing response between the treated
and the control group.
ethyl acetate/diisopropyl ether. 1H NMR (CDCl3):
d 1.08 and 1.35 [2 t,
3H þ 3H, N(CH2CH3)2], 2.25–3.53 [m, 10H, piperazine CH2’s þ 2H of
N(CH2CH3)2], 2.36 (s, 3H, NCH3), 2.96 and 3.00 [2s, 3H þ 3H,
CON(CH3)2], 3.62 [q, 2H, 2H of N(CH2CH3)2], 4.24 and 4.42 [AB q,
J ¼ 18 Hz, 1H þ 1H, HNCH2CON(CH3)2], 6.24 (br s, 1H, NH; dis-
appeared with D2O), 7.08 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼4.4 Hz, 1H, H-6),
8.15 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5), 8.72 (dd, J7,6 ¼ 4.4 Hz,
J7,5 ¼1.8 Hz, 1H, H-7); IR (KBr): 3385 (NH), 1648 s (CO), 1628 s (CO),
1598, 1551, 1515 cmꢀ1. Anal. (C22H33N7O2) C, H, N.
5.2.3. Spontaneous locomotor activity
Locomotor activity was measured by means of an activity cage
(height 35 cm, width 23 cm, depth 19 cm, Model 7401, Basile,
Comerio (VA), Italy) where the bridges the animals make or break
with their paws produce random configurations which are con-
verted into pulses. After oral administration of the compounds
under study or vehicle, mice were placed singularly into the activity
cage and locomotor activity was automatically recorded at time
intervals of 5 min for 90 min. Mice spontaneous motility was
evaluated considering the number of pulses recorded in the final
30 min period. All experiments were conducted from 9.00 to 13.00.
The percentage of inhibition was calculated from the difference
in the number of pulses between the treated and the control
group [4].
5.1.18. N,N-Diethyl-9-(dimethylamino)-5-(4-methyl-1-pipera-
zinyl)imidazo[1,2-a][1,8]naphthyridine-6-carboxamide (2n)
A mixture of 11c (4.0 mmol,1.71 g), polyphosphoric acid (0.96 g)
and POCl3 (2.0 mL) was heated at 110 ꢁC for 30 min, with stirring.
The final resinous mixture was dissolved in hot water and the
resulting dark greenish solution was made alkaline with Na2CO3
and exhaustively extracted with dichloromethane. The combined
extracts, dried over anhydrous Na2SO4 and evaporated to dryness in
vacuo, afforded a thick oil which was chromatographed on a silica
gel column eluting with the mixture dichloromethane–petroleum
ether–triethylamine (5:5:1). The eluate collected gave an oily
residue, from which, after addition of a little diisopropyl ether, pure
compound 2n separated out as a yellow crystalline solid whose
data are reported in Table 3.
5.2.4. Gastrolesivity
The acute gastrolesivity of the test compounds was evaluated by
examining the stomachs excised 5 h after oral administration of the
drugs (200 mg kg–1) in rats. The stomachs, fixed in 2% formalin
(injection of 6 mL per stomach), were opened along the greater
curvature and mounted over a flat surface. The stomachs were
5.2. Pharmacology
Wistar rats (250–300 g) and Swiss mice (25–35 g) of either sex
were used. The test compounds were suspended in 0.5% methyl-
cellulose and administered at the initial dose of 100 mg kgꢀ1 by oral
gavage (1 mL 100 gꢀ1 body weight) in animals fasted overnight.
Compounds that exhibited a statistically significant effect at this
dose were further tested at doses decreasing by a factor of two. In
these experimental tests for the evaluation of analgesic and anti-
inflammatory activity, indomethacin (10 mg kgꢀ1 oral dose) was
used as reference drug. The same dose of indomethacin was used in
rats to investigate the acute gastrolesivity while in the same test the
compounds under investigation were studied at the oral dose of
200 mg kgꢀ1. Diazepam (10 mg kgꢀ1 oral) was the reference drug in
the study of the effects of the new compounds on spontaneous
locomotor activity in mice. Groups of 8–10 animals were used and
control animals were orally treated with an equivalent volume of
vehicle alone.
examined with
a
stereomicroscope (M8 Wild Heerbrung,
Switzerland) by an observer unaware of the treatment the
rats received. Acute gastrolesivity was expressed as the number of
animals with gastric damage over the number of treated animals [4].
5.2.5. Data analysis
Results were expressed as mean ꢂ SEM. Differences between
treated and control groups were determined by Student’s t-test
(*P < 0.05 or **P < 0.01 being considered as statistically significant
or highly significant, respectively).
Acknowledgements
The Authors thank Dr. G. Domenichini for his skilful technical
assistance in pharmacological experiments, O. Gagliardo for
elemental analyses, F. Tuberoni for IR spectra, Dr. R. Raggio and
Dr. M. Anzaldi for 1H NMR spectra. The financial support from
University of Genoa and University of Parma is gratefully
acknowledged.
The ethical guidelines for investigation of experimental pain in
conscious animals were followed and all of the tests were carried
out according to the EEC ethical regulation (EEC Council 86/609;
D.L.27/01/1992, No.116).
5.2.1. Anti-inflammatory activity
Anti-inflammatory activity was studied by inducing paw
edema according to Winter’s method [10]. 1% Carrageenan
(0.1 mL) (Sigma Aldrich, Milan, Italy) was injected subcutaneously
into the plantar surface of the rat hind paw 1 h after the oral
administration of the test compound. Paw volume was deter-
mined immediately after the injection of phlogogen agent and
again 3 h later by means of a plethysmometer (Mod. 7140, Basile,
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